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G4C2 repeat expansion in C9orf72 causes the most common familial frontotemporal dementia and amyotrophic lateral sclerosis (C9FTD/ALS). The pathogenesis includes haploinsufficiency of C9orf72, which forms a protein complex with Smcr8, as well as G4C2 repeat-induced gain of function including toxic dipeptide repeats (DPRs). The key in vivo disease-driving mechanisms and how loss- and gain-of-function interplay remain poorly understood. Here, we identified dysregulation of a lysosome-ribosome biogenesis circuit as an early and key disease mechanism using a physiologically relevant mouse model with combined loss- and gain-of-function across the aging process. C9orf72 deficiency exacerbates FTD/ALS-like pathologies and behaviors in C9ORF72 bacterial artificial chromosome (C9-BAC) mice with G4C2 repeats under endogenous regulatory elements from patients. Single nucleus RNA sequencing (snRNA-seq) and bulk RNA-seq revealed that C9orf72 depletion disrupts lysosomes in neurons and leads to transcriptional dysregulation of ribosomal protein genes, which are likely due to the proteotoxic stress response and resemble ribosomopathy defects. Importantly, ectopic expression of C9orf72 or its partner Smcr8 in C9FTD/ALS mutant mice promotes lysosomal functions and restores ribosome biogenesis gene transcription, resulting in the mitigation of DPR accumulation, neurodegeneration as well as FTD/ALS-like motor and cognitive behaviors. Therefore, we conclude that loss- and gain-of-function crosstalk in C9FTD/ALS converges on neuronal dysregulation of a lysosome-ribosome biogenesis circuit leading to proteotoxicity, neurodegeneration and behavioral defects.

Community-acquired pneumonia (CAP) is a serious clinical concern. A lack of accurate diagnosis could hinder pathogen-directed therapeutic strategies. To solve this problem, we evaluated clinical application of nested multiplex polymerase chain reaction (PCR) in children with severe CAP. We prospectively enrolled 60 children with severe CAP requiring intensive care between December 2019 and November 2021 at a tertiary medical center. Nested multiplex PCR respiratory panel (RP) and pneumonia panel (PP) were performed on upper and lower respiratory tract specimens. We integrated standard-of-care tests and quantitative PCR for validation. The combination of RP, PP, and standard-of-care tests could detect at least one pathogen in 98% of cases and the mixed viral-bacterial detection rate was 65%. The positive percent agreement (PPA), and negative percent agreement (NPA) for RP were 94% and 99%; the PPA and NPA for PP were 89% and 98%. The distribution of pathogens was similar in the upper and lower respiratory tracts, and the DNA or RNA copies of pathogens in the lower respiratory tract were equal to or higher than those in the upper respiratory tract. PP detected bacterial pathogens in 40 (67%) cases, and clinicians tended to increase bacterial diagnosis and escalate antimicrobial therapy for them. RP and PP had satisfactory performance to help pediatricians make pathogenic diagnoses and establish therapy earlier. The pathogens in the upper respiratory tract had predictive diagnostic values for lower respiratory tract infections in children with severe CAP.

Dysregulation of early neurodevelopment is implicated in macrocephaly/autism disorders. However, the mechanism underlying this dysregulation, particularly in human cells, remains poorly understood. Mutations in the small GTPase gene RAB39b are associated with X-linked macrocephaly, autism spectrum disorder (ASD), and intellectual disability. The in vivo roles of RAB39b in the brain remain unknown. We generated Rab39b knockout (KO) mice and found that they exhibited cortical neurogenesis impairment, macrocephaly, and hallmark ASD behaviors, which resembled patient phenotypes. We also produced mutant human cerebral organoids that were substantially enlarged due to the overproliferation and impaired differentiation of neural progenitor cells (NPCs), which resemble neurodevelopmental deficits in KO mice. Mechanistic studies reveal that RAB39b interacts with PI3K components and its deletion promotes PI3K–AKT–mTOR signaling in NPCs of mouse cortex and cerebral organoids. The mTOR activity is robustly enhanced in mutant outer radial glia cells (oRGs), a subtype of NPCs barely detectable in rodents but abundant in human brains. Inhibition of AKT signaling rescued enlarged organoid sizes and NPC overproliferation caused by RAB39b mutations. Therefore, RAB39b mutation promotes PI3K–AKT–mTOR activity and alters cortical neurogenesis, leading to macrocephaly and autistic-like behaviors. Our studies provide new insights into neurodevelopmental dysregulation and common pathways associated with ASD across species.

Laryngeal squamous cell carcinoma (LSCC) is one of the most common laryngeal tumors, and its incidence is increasing yearly; however, whether lymph node dissection should be performed during surgery remains unclear. We retrospectively analyzed the clinical and pathological data of 246 cases of LSCC and developed a nomogram for the prediction of lymph node metastasis (LNM) of LSCC. The predictive performance and consistency of the model were evaluated using the consistency coefficient (C-index) and calibration curve, respectively. Among 246 cases of LSCC, 52 cases had metastasis with a positivity rate of 21.14%. Multivariate analyses showed that dysphagia, clinical T stage, and pathological differentiation were independent risk factors for LNM in LSCC. The accuracy of the contour map used to predict the risk for LNM was 0.809. Overall, this nomogram model can be used to evaluate LNM in patients with LSCC before surgery to decide whether to conduct neck dissection and improve patient prognosis.

Direct determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states. Here, we present PARIS2, a dramatically improved method for RNA duplex determination in vivo with >4000-fold higher efficiency than previous methods. PARIS2 captures ribosome binding sites on mRNAs, reporting translation status on a transcriptome scale. Applying PARIS2 to the U8 snoRNA mutated in the neurological disorder LCC, we discover a network of dynamic RNA structures and interactions which are destabilized by patient mutations. We report the first whole genome structure of enterovirus D68, an RNA virus that causes polio-like symptoms, revealing highly dynamic conformations altered by antiviral drugs and different pathogenic strains. We also discover a replication-associated asymmetry on the (+) and (−) strands of the viral genome. This study establishes a powerful technology for efficient interrogation of the RNA structurome and interactome in human diseases., RNA crosslinking and proximity ligation methods are used to identify transcriptome-wide base pairing interactions. Here, the authors report PARIS2 (psoralen analysis of RNA interactions and structures 2), a method for RNA duplex determination in vivo with higher efficiency than the previous PARIS method.

Introduction Traumatic brain injury (TBI) is considered as the most robust environmental risk factor for Alzheimer’s disease (AD). Besides direct neuronal injury and neuroinflammation, vascular impairment is also a hallmark event of the pathological cascade after TBI. However, the vascular connection between TBI and subsequent AD pathogenesis remains underexplored. Methods In a closed-head mild TBI (mTBI) model in mice with controlled cortical impact, we examined the time courses of microvascular injury, blood–brain barrier (BBB) dysfunction, gliosis and motor function impairment in wild type C57BL/6 mice. We also evaluated the BBB integrity, amyloid pathology as well as cognitive functions after mTBI in the 5xFAD mouse model of AD. Results mTBI induced microvascular injury with BBB breakdown, pericyte loss, basement membrane alteration and cerebral blood flow reduction in mice, in which BBB breakdown preceded gliosis. More importantly, mTBI accelerated BBB leakage, amyloid pathology and cognitive impairment in the 5xFAD mice. Discussion Our data demonstrated that microvascular injury plays a key role in the pathogenesis of AD after mTBI. Therefore, restoring vascular functions might be beneficial for patients with mTBI, and potentially reduce the risk of developing AD.

SUMMARYDirect determination of RNA structures and interactions in living cells is critical for understanding their functions. Current crosslinking and proximity-ligation approaches are fundamentally limited due to inefficient RNA crosslinking, purification and high-level photochemical damages. Here we present PARIS2 (psoralen analysis of RNA interactions and structures, second generation), a re-invented method for capturing RNA duplexes in cells with three orders of magnitude improved efficiency. PARIS2 captures ribosome small subunit (SSU) binding sites on mRNAs, reporting translation status on a transcriptome wide scale, and captures spliceosomal snRNP binding sites on various RNA targets. We determine the RNA genome structure of enterovirus D68, a re-emerging viral pathogen associated with severe neurological symptoms, and discover alternative conformations in the internal ribosome entry site (IRES) that controls translation initiation. Together, these results reveal new aspects of RNA photochemistry and enzymology, and enable highly efficient interrogation of the RNA structurome and interactome in cells.

IntroductionTraumatic brain injury (TBI) is considered as the most robust environmental risk factor for Alzheimer’s disease (AD). Besides direct neuronal injury and neuroinflammation, vascular impairment is also a hallmark event of the pathological cascade after TBI. However, the vascular connection between TBI and subsequent AD pathogenesis remains underexplored.MethodsWe established a closed-head mild TBI (mTBI) model in mice with controlled cortical impact, and examined the time courses of microvascular injury, blood-brain barrier (BBB) dysfunction, gliosis and motor function impairment in wild type C57BL/6 mice. We also determined the brain clearance of β-amyloid, as well as amyloid pathology and cognitive functions after mTBI in the 5xFAD mouse model of AD.ResultsmTBI induced microvascular injury with BBB breakdown, pericyte loss and cerebral blood flow reduction in mice, which preceded gliosis. mTBI also impaired brain amyloid clearance via the vascular pathways. More importantly, mTBI accelerated amyloid pathology and cognitive impairment in the 5xFAD mice.DiscussionOur data demonstrated that microvascular injury plays a key role in the pathogenesis of AD after mTBI. Therefore, restoring vascular functions might be beneficial for patients with mTBI, and potentially reduce the risk of developing AD.

How lysosome and MTORC1 signaling interact remains elusive in terminally differentiated cells. A G4C2 repeat expansion in C9orf72 is the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS-FTD). We previously identified a C9orf72-SMCR8-containing complex. Here we found that c9orf72 and smcr8 double-knockout (dKO) mice exhibit similar but more severe immune defects than the individual knockouts. In c9orf72 or smcr8 mutant macrophages, lysosomal degradation and exocytosis were impaired due to the disruption of autolysosome acidification. As a result of impaired lysosomal degradation, MTOR protein was aberrantly increased, resulting in MTORC1 signaling overactivation. Inhibition of hyperactive MTORC1 partially rescued macrophage dysfunction, splenomegaly and lymphadenopathy in c9orf72 or smcr8 mutant mice. Pharmacological inhibition of lysosomal degradation upregulated MTOR protein and MTORC1 signaling in differentiated wild-type macrophages, which resemble phenotypes in KO mice. In contrast, C9orf72 or Smcr8 depletion in proliferating macrophages decreased MTORC1 signaling. Our studies causatively link C9orf72-SMCR8’s cellular functions in lysosomal degradation, exocytosis, and MTORC1 signaling with their organism-level immune regulation, suggesting cell state (proliferation vs. differentiation)-dependent regulation of MTOR signaling via lysosomes.Abbreviations: ALS: amyotrophic lateral sclerosis; ATG13: autophagy related 13; BMDMs: bone marrow-derived macrophages; BafA(1): bafilomycin A(1); C9orf72: C9orf72, member of C9orf72-SMCR8 complex; CD68: CD68 antigen; ConA: concanamycin A; dKO: double knockout; DENN: differentially expressed in normal and neoplastic cells; FTD: frontotemporal dementia; GEF: guanine nucleotide exchange factor; IFNB1: interferon beta 1, fibroblast; IFNG: interferon gamma; IL1B/IL-1β: interleukin 1 beta; IL6: interleukin 6; iPSCs: induced pluripotent stem cells; LAMP1: lysosomal-associated membrane protein 1; LPOs: LAMP1-positive organelles; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; LPS: lipopolysaccharide; MTORC1: mechanistic target of rapamycin kinase complex 1; MEFs: mouse embryonic fibroblasts; MNs: motor neurons; NOS2/iNOS: nitric oxide synthase 2, inducible; RAN: repeat-associated non-AUG; RB1CC1/FIP200: RB1-inducible coiled-coil 1; RPS6/S6: ribosomal protein S6; RPS6KB1/S6K1: ribosomal protein S6 kinase, polypeptide 1; SMCR8: Smith-Magenis syndrome chromosome region, candidate 8; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TNF: tumor necrosis factor; TSC1: TSC complex subunit 1; ULK1: unc-51 like kinase 1; v-ATPase: vacuolar-type H⁺-translocating ATPase

Primary microcephaly is caused by mutations in genes encoding centrosomal proteins including WDR62 and KIF2A. However, mechanisms underlying human microcephaly remain elusive. By creating mutant mice and human cerebral organoids, here we found that WDR62 deletion resulted in a reduction in the size of mouse brains and organoids due to the disruption of neural progenitor cells (NPCs), including outer radial glia (oRG). WDR62 ablation led to retarded cilium disassembly, long cilium, and delayed cell cycle progression leading to decreased proliferation and premature differentiation of NPCs. Mechanistically, WDR62 interacts with and promotes CEP170’s localization to the basal body of primary cilium, where CEP170 recruits microtubule-depolymerizing factor KIF2A to disassemble cilium. WDR62 depletion reduced KIF2A’s basal body localization, and enhanced KIF2A expression partially rescued deficits in cilium length and NPC proliferation. Thus, modeling microcephaly with cerebral organoids and mice reveals a WDR62-CEP170-KIF2A pathway promoting cilium disassembly, disruption of which contributes to microcephaly., Mutations in WDR62 are the second most common genetic cause of autosomal recessive primary microcephaly, yet the molecular mechanisms underlying this pathogenesis remain unclear. Here, authors demonstrate that WDR62 depletion leads to neural precursor cell depletion and microcephaly via WDR62-CEP170-KIF2A pathway that promotes cilium disassembly.

Neural progenitor cells (NPCs) undergo rapid proliferation during neurulation. This rapid growth generates a high demand for mRNA translation in a timing-dependent manner, but its underlying mechanism remains poorly understood. Lin28 is an RNA-binding protein with two paralogs, Lin28a and Lin28b, in mammals. Mice with Lin28b deletion exhibit no developmental defects, whereas we previously reported that Lin28a deletion led to microcephaly. Here we found that Lin28a/b double knockout (dKO) mice displayed neural tube defects (NTDs) coupled with reduced proliferation and precocious differentiation of NPCs. Using ribosomal protein 24 hypomorphic mice (Rpl24Bst/+) as a genetic tool to dampen global protein synthesis, we found that Lin28a−/−;Rpl24Bst/+ compound mutants exhibited NTDs resembling those seen in Lin28a/b dKO mice. Increased NPC numbers and brain sizes in Lin28a-overexpressing mice were rescued by Rpl24Bst/+ heterozygosity. Mechanistically, polysome profiling revealed reduced translation of genes involved in the regulation of cell cycle, ribosome biogenesis, and translation in dKO mutants. Ribosome biogenesis was reduced in dKO and increased in Lin28a-overexpressing NPCs. Therefore, Lin28-mediated promotion of protein synthesis is essential for NPC maintenance and early brain development.

Craniosynostosis results from premature fusion of the cranial suture(s), which contain mesenchymal stem cells (MSCs) that are crucial for calvarial expansion in coordination with brain growth. Infants with craniosynostosis have skull dysmorphology, increased intracranial pressure, and complications such as neurocognitive impairment that compromise quality of life. Animal models recapitulating these phenotypes are lacking, hampering the development of urgently needed innovative therapies. Here we show that Twist1(+/−) mice with craniosynostosis have increased intracranial pressure and neurocognitive behavioral abnormalities, recapitulating features of human Saethre-Chotzen syndrome. Using a biodegradable material combined with MSCs, we have successfully regenerated a functional cranial suture that corrects skull deformity, normalizes intracranial pressure and rescues neurocognitive behavior deficits. The regenerated suture creates a niche into which endogenous MSCs migrated, sustaining calvarial bone homeostasis and repair. MSC-based cranial suture regeneration offers a paradigm shift in treatment to reverse skull and neurocognitive abnormalities in this devastating disease.

Summary Zika virus (ZIKV) causes microcephaly and disrupts neurogenesis. Dicer-mediated miRNA biogenesis is required for embryonic brain development and has been suggested to be disrupted upon ZIKV infection. Here we mapped the ZIKV-host interactome in neural stem cells (NSCs) and found that Dicer is specifically targeted by the capsid from ZIKV, but not other flaviviruses, to facilitate ZIKV infection. We identified a capsid mutant (H41R) that loses this interaction and does not suppress Dicer activity. Consistently, ZIKV-H41R is less virulent and does not inhibit neurogenesis in vitro or corticogenesis in utero. Epidemic ZIKV strains contain capsid mutations that increase Dicer binding affinity and enhance pathogenicity. ZIKV-infected NSCs show global dampening of miRNA production, including key miRNAs linked to neurogenesis, which is not observed after ZIKV-H41R infection. Together these findings show that capsid-dependent suppression of Dicer is a major determinant of ZIKV immune evasion and pathogenesis and may underlie ZIKV-related microcephaly.

An investigation on the structural, electronic, thermodynamic, and mechanical properties of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX)/2-methylpyridine-N-oxide (MPNO) cocrystal was carried out from 0 to 100 GPa by using a dispersion-corrected density functional theory (DFT-D) method. Our calculated crystal structure is in excellent agreement with experimental results at ambient pressure. Based on the analysis of lattice parameters, lattice angles, bond lengths, bond angles, and dihedral angles under high pressure, we observe that HMX molecules in the cocrystal bulk are seriously distorted but MPNO molecules remain relatively unchanged. Hydrogen bond lengths are greatly shortened under high pressure. In addition, with the increase in pressure, the bandgap decreases gradually. However, it increases suddenly at 70 GPa. Some important hydrogen bonds between HMX and MPNO are also observed in the density of states spectrum. According to the thermodynamic analysis, this cocrystal is more easi...

A novel 1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX)/pyridine-N-oxide (PNO) cocrystal at 1:1 molar ratio was synthesized by a solvent evaporation method, and its crystal structure was determined using X-ray diffraction (XRD). It crystallizes in the orthorhombic system with the Pbcn space group and cell parameters a = 12.712(3)A, b = 9.315(3)A, c = 12.909(3)A. In addition, detonation performance of this cocrystal was estimated. The predicted detonation velocity and detonation pressure of this cocrystal are 7.47 km/s and 23.20 GPa, respectively, suggesting that it is less powerful than β-HMX. Finally, density functional theory, involving binding energy, atoms in molecule (AIM) theory, natural bond orbital (NBO) analysis, band structure, and density of states, was adopted to characterize the driving forces for the formation of this cocrystal. The results show that driving forces are dominated by the interactions between O atoms of PNO and methylene groups of HMX. It is expected that this research pro...

G4C2 repeat expansions in an intron of C9ORF72 cause the most common familial amyotrophic lateral sclerosis and frontotemporal dementia (collectively, C9ALS/FTD). Mechanisms and mediators of C9ALS/FTD pathogenesis remain poorly understood. C9orf72 and Smcr8 form a protein complex. Here, we show that expression of Smcr8, like C9orf72, is reduced in C9ALS/FTD mouse models and patient tissues. Since Smcr8 is highly conserved between human and mouse, we evaluated the effects of Smcr8 downregulation in mice. Smcr8 knockout (KO) mice exhibited motor behavior deficits, which resemble those of C9ALS/FTD mouse models, and displayed axonal swellings in their spinal cords and neuromuscular junctions. These deficits are caused by impaired autophagy-lysosomal functions due to disrupted axonal transport in mutant motor neurons. Consistent with its interaction with C9orf72 and their downregulation in patient tissues, Smcr8 deficiency exacerbated autophagy-lysosomal impairment in C9orf72 KO mice. The disease relevance of Smcr8 downregulation was reflected by exacerbated axonal swellings and gain of toxicity pathology arising from Smcr8 haploinsufficiency in a mouse model of C9ALS/FTD. Thus, our in vivo studies suggested that Smcr8 deficiency impairs axonal transport dependent autophagy-lysosomal function and exacerbates axonal degeneration and gain of toxicity in C9ALS/FTD mouse models.

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Differentiation of smooth muscle cells (SMCs) is critical for proper vasculogenesis and angiogenesis. However, the molecular mechanisms controlling SMC differentiation are not completely understood. During embryogenesis, the transcription factor mesenchyme homeobox 1 (Meox1) is expressed in the early developing somite, which is one of the origins of SMCs. In the present study, we identified Meox1 as a positive regulator of SMC differentiation. We found that transforming growth factor-β (TGF-β) induces Meox1 expression in the initial phase of SMC differentiation of pluripotent murine C3H10T1/2 cells. shRNA-mediated Meox1 knockdown suppressed TGF-β–induced expression of SMC early markers, whereas Meox1 overexpression increased expression of these markers. Mechanistically, Meox1 promoted SMAD family member 3 (Smad3) nuclear retention during the early stage of TGF-β stimulation because Meox1 inhibited protein phosphatase Mg(2+)/Mn(2+)-dependent 1A (PPM1A) and thereby prevented PPM1A-mediated Smad3 dephosphorylation. Meox1 appears to promote PPM1A degradation, leading to sustained Smad3 phosphorylation, thus allowing Smad3 to stimulate SMC gene transcription. In vivo, Meox1 knockdown in mouse embryos impaired SMC marker expression in the descending aorta of neonatal mice, indicating that Meox1 is essential for SMC differentiation during embryonic development. In summary, the transcriptional regulator Meox1 controls TGF-β–induced SMC differentiation from mesenchymal progenitor cells by preventing PPM1A-mediated Smad3 dephosphorylation, thereby supporting SMC gene expression.

The cardiovascular system develops during the early stages of embryogenesis, and differentiation of smooth muscle cells (SMCs) is essential for that process. SMC differentiation is critically regulated by transforming growth factor (TGF)-β/SMAD family member 3 (SMAD3) signaling, but other regulators may also play a role. For example, long noncoding RNAs (lncRNAs) regulate various cellular activities and events, such as proliferation, differentiation, and apoptosis. However, whether long noncoding RNAs also regulate SMC differentiation remains largely unknown. Here, using the murine cell line C3H10T1/2, we found that brain cytoplasmic RNA 1 (BC1) is an important regulator of SMC differentiation. BC1 overexpression suppressed, whereas BC1 knockdown promoted, TGF-β-induced SMC differentiation, as indicated by altered cell morphology and expression of multiple SMC markers, including smooth muscle α-actin (αSMA), calponin, and smooth muscle 22α (SM22α). BC1 appeared to block SMAD3 activity and inhibit SMC marker gene transcription. Mechanistically, BC1 bound to SMAD3 via RNA SMAD-binding elements (rSBEs) and thus impeded TGF-β–induced SMAD3 translocation to the nucleus. This prevented SMAD3 from binding to SBEs in SMC marker gene promoters, an essential event in SMC marker transcription. In vivo, BC1 overexpression in mouse embryos impaired vascular SMC differentiation, leading to structural defects in the artery wall, such as random breaks in the elastic lamina, abnormal collagen deposition on SM fibers, and disorganized extracellular matrix proteins in the media of the neonatal aorta. Our results suggest that BC1 is a suppressor of SMC differentiation during vascular development.

Neural progenitor cells (NPCs) have distinct proliferation capacities at different stages of brain development. Lin28 is an RNA-binding protein with two homologs in mice: Lin28a and Lin28b. Here we show that Lin28a/b are enriched in early NPCs and their expression declines during neural differentiation. Lin28a single-knockout mice show reduced NPC proliferation, enhanced cell cycle exit and a smaller brain, whereas mice lacking both Lin28a alleles and one Lin28b allele display similar but more severe phenotypes. Ectopic expression of Lin28a in mice results in increased NPC proliferation, NPC numbers and brain size. Mechanistically, Lin28a physically and functionally interacts with Imp1 (Igf2bp1) and regulates Igf2-mTOR signaling. The function of Lin28a/b in NPCs could be attributed, at least in part, to the regulation of their mRNA targets that encode Igf1r and Hmga2. Thus, Lin28a and Lin28b have overlapping functions in temporally regulating NPC proliferation during early brain development.

The Zika virus (ZIKV) has two lineages, Asian and African, and their impact on developing brains has not been compared. Dengue virus (DENV) is a close family member of ZIKV and co-circulates with ZIKV. Here we performed intracerebral inoculation of embryonic mouse brains with dengue virus 2 (DENV2), and found that DENV2 is sufficient to cause smaller brain size due to increased cell death in neural progenitor cells (NPCs) and neurons. Compared to the currently circulating Asian lineage of ZIKV (MEX1-44), DENV2 grows slower, causes less neuronal death, and fails to cause postnatal animal death. Surprisingly, our side-by-side comparison uncovered that African ZIKV isolate (MR-766) is more potent in causing brain damage and postnatal lethality than MEX1-44. In comparison to MEX1-44, MR-766 grows faster in NPCs and in the developing brain, and causes more pronounced cell death in NPCs and neurons, resulting in more severe neuronal loss. Together, these results reveal that DENV2 is sufficient to cause smaller brain sizes, and suggest that the ZIKV African lineage is more virulent and causes more severe brain damage than the Asian lineage.

Nowadays, the instant beverage has been a necessary for daily life. The chain stores of instant beverages are popular in recent years. The sales volume per year of instant beverages reaches to US$ 133 million in Taiwan. The reason of the prosperity of the beverages in Taiwan is that the beverages are able to be made according to custom’s needs. So that the beverage can be fit to the custom’s favorite and habit. Therefore, for the considerations of safety, sanitary, and consistent quality, an instant beverage maker is needed to be developed. This study applied a systematical design procedure to develop and design the beverage maker. Firstly, marketing and technical survey, user needs and ergonomic analysis, and materials and methods of beverages investigation are conducted to determine the design requirements and specification of the product. Secondly, a series of design processes are proceed to develop the product, among them are concept design, mechanism design, ergonomic design, appearance design, 3D modeling and model making and testing. Finally, a new product is designed with the following features: (1) the instant beverage can be produced to match the favorite and habit of the customs; (2) the beverage can be produced with a high quality and stability, (3) the beverage maker with automatic processing functions thus saving manpower and manufacturing time; (4) this product with a friendly interface thus facilitating users to operate and clean it easily; and (5) the appearance is appealed and has high-tech and professional image. The newly designed product can be sold in Taiwan and the international markets. The design process and results of the study can be used a reference for product design and development.

Failure of embryonic neural tube closure results in the second most common class of birth defects known as neural tube defects (NTDs). While NTDs are likely the result of complex multigenic dysfunction, it is not known whether polymorphisms in epigenetic regulators may be risk factors for NTDs. Here we characterized Baf155msp3, a unique ENU-induced allele in mice. Homozygous Baf155mps3 embryos exhibit highly penetrant exencephaly, allowing us to investigate the roles of an assembled, but malfunctional BAF chromatin remodeling complex in vivo at the time of neural tube closure. Evidence of defects in proliferation and apoptosis were found within the neural tube. RNA-Seq analysis revealed that surprisingly few genes showed altered expression in Baf155 mutant neural tissue, given the broad epigenetic role of the BAF complex, but included genes involved in neural development and cell survival. Moreover, gene expression changes between individual mutants were variable even though the NTD was consistently observed. This suggests that inconsistent gene regulation contributes to failed neural tube closure. These results shed light on the role of the BAF complex in the process of neural tube closure and highlight the importance of studying missense alleles to understand epigenetic regulation during critical phases of development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 483–497, 2014

Zika virus (ZIKV) infection of pregnant women can result in fetal brain abnormalities. It has been established that ZIKV disrupts neural progenitor cells (NPCs) and leads to embryonic microcephaly. However, the fate of other cell types in the developing brain and their contributions to ZIKV-associated brain abnormalities remain largely unknown. Using intracerebral inoculation of embryonic mouse brains, we found that ZIKV infection leads to postnatal growth restriction including microcephaly. In addition to cell cycle arrest and apoptosis of NPCs, ZIKV infection causes massive neuronal death and axonal rarefaction which phenocopy fetal brain abnormalities in humans. Importantly, ZIKV infection leads to abnormal vascular density and diameter in the developing brain, resulting in a leaky blood-brain-barrier (BBB). Massive neuronal death and BBB leakage indicate brain damage, which is further supported by extensive microglial activation and astrogliosis in virally infected brains. Global gene analyses reveal dysregulation of genes associated with immune responses in virus-infected brains. Thus, our data suggest that ZIKV triggers a strong immune response and disrupts neurovascular development, resulting in postnatal microcephaly with extensive brain damage.

The objective of this paper is to present a new hybrid predictive current-mode control for power factor corrector with on-line parameter tuning. The proposed method can effectively solve the problems caused by inductance variation due to manufacturing tolerances, load current, magnetic intensity and temperature etc. for the power factor corrector with hybrid predictive current-mode control. The inductance variation may result in the predicted duty error and conduction mode switching error. Analysis of predicted duty error and conduction mode switching error contributed by the inductance variation for hybrid predictive current-mode control will be presented. The conduction mode switching error and duty error will deteriorate the power rating and reduce the efficiency. An on-line inductance tuning method for the power factor corrector with hybrid predictive current-mode control will be proposed in this paper. And this tuned parameter is used to correct the predicted duty error. The specifications of the converter include: input voltage 100 V/60 Hz, output voltage 380 VDC, output power 760 W, switching frequency 65 kHz. The control software is developed and verified using a microprocessor, TMS320F28335. Details of the controller design, simulation and experimental results are presented to fully support the theoretical analysis and validate the proposed method.

A dual role of the C9ORF72/SMCR8-containing complex in autophagy initiation and autophagic flux., The intronic GGGGCC hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) is a prevalent genetic abnormality identified in both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Smith-Magenis syndrome chromosomal region candidate gene 8 (SMCR8) is a protein with unclear functions. We report that C9ORF72 is a component of a multiprotein complex containing SMCR8, WDR41, and ATG101 (an important regulator of autophagy). The C9ORF72 complex displays guanosine triphosphatase (GTPase) activity and acts as a guanosine diphosphate–guanosine 5′-triphosphate (GDP-GTP) exchange factor (GEF) for RAB39B. We created Smcr8 knockout mice and found that Smcr8 mutant cells exhibit impaired autophagy induction, which is similarly observed in C9orf72 knockdown cells. Mechanistically, SMCR8/C9ORF72 interacts with the key autophagy initiation ULK1 complex and regulates expression and activity of ULK1. The complex has an additional role in regulating later stages of autophagy. Whereas autophagic flux is enhanced in C9orf72 knockdown cells, depletion of Smcr8 results in a reduced flux with an abnormal expression of lysosomal enzymes. Thus, C9ORF72 and SMCR8 have similar functions in modulating autophagy induction by regulating ULK1 and play distinct roles in regulating autophagic flux.

Rationale: Mammalian heart has minimal regenerative capacity. In response to mechanical or pathological stress, the heart undergoes cardiac remodeling. Pressure and volume overload in the heart cause increased size (hypertrophic growth) of cardiomyocytes. Whereas the regulatory pathways that activate cardiac hypertrophy have been well-established, the molecular events that inhibit or repress cardiac hypertrophy are less known. Objective: To identify and investigate novel regulators that modulate cardiac hypertrophy. Methods and Results: Here, we report the identification, characterization, and functional examination of a novel cardiac Isl1-interacting protein (CIP). CIP was identified from a bioinformatic search for novel cardiac-expressed genes in mouse embryonic hearts. CIP encodes a nuclear protein without recognizable motifs. Northern blotting, in situ hybridization, and reporter gene tracing demonstrated that CIP is highly expressed in cardiomyocytes of developing and adult hearts. Yeast two-hybrid screening identified Isl1, a LIM/homeodomain transcription factor essential for the specification of cardiac progenitor cells in the second heart field, as a cofactor of CIP. CIP directly interacted with Isl1, and we mapped the domains of these two proteins, which mediate their interaction. We show that CIP represses the transcriptional activity of Isl1 in the activation of the myocyte enhancer factor 2C. The expression of CIP was dramatically reduced in hypertrophic cardiomyocytes. Most importantly, overexpression of CIP repressed agonist-induced cardiomyocyte hypertrophy. Conclusions: Our studies therefore identify CIP as a novel regulator of cardiac hypertrophy.

According to the work requirements of a production line of sheet metal handling, using the mechanical design principles and methods, pneumatic technology and PLC control technology for sheet metal handling manipulator research and design, and development a style of automatic handling manipulator meeting the requirements. This article introduced the sheet metal handling manipulator and principles of the basic structure of the hand, pneumatic systems and PLC control system design and analysis. Practice shows that the manipulator has a simple structure, reliable, efficient features, which meet actual production needs.